We propose to utilize advanced EPR methods to characterize a key intermediate in dioxygen reduction in the terminal respiratory enzyme, cytochrome c oxidase. Recent resonance Raman experiments have demonstrated that the O-O bond is broken at the so-called "peroxy" intermediate level. Of the four electrons required for this reduction chemistry, two could readily result from oxidation of the heme a3 Fe from Fe(II) to Fe(IV), and one from the oxidation of CuB from Cu(I) to Cu(II). Recent x-ray structures of cytochrome c oxidase have shown the presence of a modified tyrosine, covalently crosslinked to a histidine residue, positioned so as to hydrogen-bond with the dioxygen substrate bound in the heme a3-CuB catalytic center of the enzyme. This has led to the postulate that the fourth electron is provided by the modified tyrosine residue, with hydrogen atom transfer from this amino acid providing the trigger for O-O bond cleavage [Proshlyakov DA, Pressler MA & Babcock GT (1998) Proc. Natl. Acad. Sci USA 95:8020-8025]. The resultant "peroxy" intermediate state can be trapped following O2 addition to the "mixed valence" form of the enzyme, where only the heme a3 and CuB centers are reduced, or by H2O2 addition to the oxidized form of the enzyme. This state has an even number of unpaired electrons, and we propose to utilize parallel polarization EPR spectroscopy to detect integer spin transitions from the catalytic domain of cytochrome c oxidase trapped in this intermediate state. Analysis of the resultant spectra should provide extremely useful information concerning the chemical nature of this "peroxy" intermediate. In addition, we will employ time-resolved EPR spectroscopy to test whether transient radicals are formed following CO-photolysis of the reduced enzyme in the presence of O2. These experiments hold much promise for providing key spectroscopic information concerning the mechanism of dioxygen reduction and its coupling to proton pumping in this crucial mitochondrial enzyme.